Ultra-High Resolution Analysis of Genomic Alterations in High-Risk Acute Lymphoblastic Leukemia.

Previous genomic analyses identified a high frequency of genetic alterations targeting B-lymphoid development (>40%) and cell cycle/tumor suppression (CDKN2A/B, 35%) in pediatric B-progenitor acute lymphoblastic leukemia (B-ALL). The frequency and nature of copy number alterations (CNAs) differs significantly across ALL subtype with less than one CNA/case in MLL ALL and over 8 lesions/case in BCR-ABL1 and ETV6-RUNX1 ALL; and the near obligate presence of IKZF1 (Ikaros) deletion in BCR-ABL1 ALL, but never in ETV6-RUNX1 ALL. Although important insights have been obtained from these genome-wide CNAs, it is important to realize that the SNP array platforms that have been used are limited by uneven probe spacing and poor coverage of some genes (including IKZF1 and CDKN2A/B). Moreover, additional CNAs are detected as array resolution increases (e.g. 6.5 lesions per BCR-ABL1 case using 315,000 markers, and 8.8 for 615,000 marker data). Thus, the true frequency of CNAs in ALL remains unknown. To address this question, we now report ultra-high resolution CNA analysis using 2.17 million feature oligonucleotide arrays (Roche Nimblegen) of 20 MLL, 20 BCR-ABL1, and 4 miscellaneous karyotype B-ALL cases. All had previously been examined using Affymetrix 500K SNP arrays. We identified a mean of 6.2 deletions (range 0–38) and 1.4 gains (0–13) per case. There were more lesions in BCR-ABL1 than MLL ALL cases (mean 11.2 v 1.15 deletions, P<0.0001; 2.3 v 0.45 amplifications, P=0.01). Notably, more lesions were detected by this 2.17 million feature array in comparison to previous Affymetrix SNP array data in BCRABL1 ALL (13.5 v 8.8 lesions/case, P=0.001) but not MLL ALL (1.6 v 1.3 lesions/case, P=NS). Moreover, the Nimblegen platform robustly identified focal deletions in genes poorly covered by SNP arrays (IKZF1, CDKN2A/B) and detected additional deletions not previously identified (e.g. deletion of HBS1L immediately adjacent to MYB). The most common lesions in ALL were deletions of IKZF1 (80% BCR-ABL1, none in MLL), CDKN2A/B (60% BCR-ABL1, 20% MLL) and CNAs of PAX5 (50% BCR-ABL1, 20% MLL). High array resolution enabled PCR-based mapping and sequencing of the genomic breakpoints of multiple recurring deletions (e.g. ADD3, BTLA, CDKN2A/B, C20orf94, HBS1L, IKZF1 and PAX5), each of which bore hallmarks of aberrant RAG-mediated recombination.

Due to the small size of the CDKN2A/B genomic locus (41.5 Kb), previous genomic analyses have been unable to accurately determine the frequency of deletion of each of the three tumor suppressors (INK4A, ARF, and INK4B) encoded by this locus. We performed genomic quantitative PCR (gqPCR) across the locus (CDKN2A exons 1b, 1a and 2; CDKN2B exons 1 and 2), which revealed complete concordance between deletions identified by the Nimblegen array and confirmatory gqPCR. However, gqPCR was required to precisely delineate the extent of deletion in each case. All deletions involved both CDKN2A and CDKN2B loci except one MLL case (CDKN2A only), and the same degree of deletion (mono- or bi-allelic) was usually uniform across the locus for each case. Exceptions included two cases harboring homozygous deletions of INK4B and ARF but hemizygous deletion of INK4A, and a case with deletion of ARF-INK4A but not INK4B. To extend this analysis to epigenetic silencing of CDKN2A/B, we performed quantitative methylation analysis of six CDKN2A and two CDKN2B CpG islands by MALDI-TOF mass spectrometry for all BCR-ABL1 cases and 18 CDKN2A/B non-deleted MLL, ETV6-RUNX, and T-ALL cases. Strikingly, whereas high level CpG methylation of CDKN2B and CDKN2A exons 2 and 3 was observed in half the MLL and T-ALL cases, no high level methylation was observed in BCR-ABL1 ALL.

These data demonstrate that identification of all CNAs in BCR-ABL1 ALL is critically dependent on array resolution, whereas MLL -rearranged leukemias harbor very few CNAs. Importantly, the use of high density arrays with even probe distribution improves the ability to robustly identify focal CNAs in genes poorly covered by SNP arrays, and to further characterize the mechanism of deletion by sequencing. Furthermore, even with the highest resolution arrays, complementary quantitative PCR across CDKN2A/B is required to precisely define the extent of deletion of this compact gene cluster. Thus, oligonucleotide array or sequencing-based platforms allowing true tiling across the genome will be required to determine the full complement of CNAs in ALL.